Non-pathogenic microbiota accelerate age-related CpG Island methylation in colonic mucosa

Abstract

DNA methylation is an epigenetic process altered in cancer and ageing. Age-related methylation drift can be used to estimate lifespan and can be influenced by extrinsic factors such as diet. Here, we report that non-pathogenic microbiota accelerate age-related methylation drift in the colon when compared with germ-free mice. DNA methylation analyses showed that microbiota and IL10KO were associated with changes in 5% and 4.1% of CpG sites, while mice with both factors had 18% alterations. Microbiota, IL10KO, and their combination altered 0.4%, 0.4%, and 4% of CpG island methylation, respectively. These are comparable to what is seen in colon cancer. Ageing changes were accelerated in the IL10KO mice with microbiota, and the affected genes were more likely to be altered in colon cancer. Thus, the microbiota affect DNA methylation of the colon in patterns reminiscent of what is observed in ageing and colorectal cancer.

Keywords: Ageing; DNA methylation; Germ-free; Inflammation; Microbiota.

Figure 1.

Microbiota influence DNA methylation. a-c) Volcano plot analysis showing methylation differences between SPF and GF mice (a), Il10^−/− and GF mice (b), and SPF-Il10^−/− and GF mice (c). The x-axis shows the difference in average methylation between SPF and GF mice for a given site. The y-axis is the negative log(10) of the p-value, which was determined with a Student’s t-test. All sites above the dotted line are significant at p ≤ 0.05. Green sites change at a magnitude of 5% or greater. d-f) Bar graphs showing the proportion and type of CpG sites that change at least 5% between SPF and GF mice (d), Il10^−/− and GF mice (e), and SPF-Il10^−/− and GF mice (f). Shore indicates sites that are not in CGIs but within 2,000 bp of them. (g) Bar graph showing the proportion of CpG sites that show DNA methylation alterations in the volcano plots in a-c. (h) Bar graph showing the proportion of CpG sites within CpG islands that show DNA methylation alterations in the volcano plots in a-c.

Figure 2.

AOM is a potent hypomethylating carcinogen. (a) Volcano plot analysis showing methylation differences between SPF and SPF+AOM mice. See Figure 1 for graph details. (b) Bar graph showing the proportion and type of CpG sites that change at least 5% in (a). (c) Volcano plot analysis showing methylation differences between SPF and SPF-Il10^−/− mice. See Figure 1 for graph details. (d) Bar graph showing the proportion and type of CpG sites that change at least 5% in (c).

Figure 3.

Linear regression model of the effects of microbiota, Il10^−/−and AOM on DNA methylation. (a) Shows Volcano plots of FDR (0.05) corrected significant methylation changes attributed to each external exposure. The x-axis indicates the linear model slope for individual CpG sites (equivalent to % change in methylation) while the y-axis is the negative log(10) of the q-value. (b) Shows Upset plots of shared/unique hypomethylation events while (c) shows UpSet plots of shared/unique hypermethylation events. The number of shared or uniquely altered CpG sites is indicated on top of each vertical bar.

Figure 4.

Microbiota and inflammation modify the same CpG sites subject to age-related methylation drift. (a) Volcano plot analysis showing methylation differences between young and old mice. See Figure 1a for graph details. (b) UpSet plots of shared/unique hypomethylation events between sites that decrease at least 5% during ageing and in the different exposures analysed in Figures 1–3. ‘Inflammation’ refers to Il10^−/−/SPF mice. (c) Same analysis as in B for sites that increase methylation at least 5%. (d) Scatter plot of average methylation change with age (x-axis) to average change by exposures (y-axis) for all sites that change at least 5% with age. Pearson r, p-value, and slope are indicated in each plot.

Figure 5.

Genes affected by extrinsic exposures are more likely to be altered in cancer. (a) Scatter plot of DNA methylation change by ageing or exposure (x-axis) compared to DNA methylation change in colon cancer TCGA samples. (b) Odds ratios compute enrichment of genes altered by ageing or different extrinsic exposures among genes altered in colon cancer (TCGA data). The odds ratios are computed separately for gene promoters hypomethylated (left) or hypermethylated (right) by exposures and in colon cancer.